Total synthesis of chondramide C and its binding mode to F-actin

Article abstract of DOI:10.1002/anie.200801010

(Chemical Equation Presented) Actin glue: An E-selective ring-closing metathesis as the key step allowed the solid-phase-based total synthesis of the F-actin stabilizer chondramide C as well as the establishment of its hitherto unknown stereochemistry. A strong influence of the polyketide configuration was revealed in cellular assays. Docking studies on the F-actin filament structure led to a detailed model of the binding site.

Full text of DOI:10.1002/anie.200801010

Angewandte
Chemie
DOI: 10.1002/anie.200801010
Drug Design
Total Synthesis of Chondramide C and Its Binding Mode to F-Actin**
Herbert Waldmann,* Tai-Shan Hu, Steffen Renner, Sascha Menninger, RenØ Tannert,
Toshiro Oda, and Hans-Dieter Arndt*
Dedicated to Professor Reinhard W.Hoffmann on the ooccasion of his 75th birthday
The actin cytoskeleton maintains the cellꢀs shape and is
essential for cell movement, phagocytosis, and cytokinesis.[1]
Small molecules that interfere with the dynamic assembly and
disassembly of actin have hence proven to be invaluable tools
for chemical biology and medicinal chemistry research.[1a,2] In
particular, natural products have been uncovered that either
inhibit or induce F-actin polymer formation from the mono-
meric G-actin and thereby modulate the maintenance of the
cytoskeleton.[1a,2]
Phalloidin (1),[3] jasplakinolide (2),[4] and chondramide C
(3)[5] (Scheme 1) stabilize F-actin by a similar mode of
action.[6] In contrast to 1,[7] jasplakinolide (2) and chondra-
mide C (3) are cell-permeable and display potency against
tumor cell lines which renders them interesting target
structures for drug discovery. While several total syntheses
of 2 have been described,[8] the commercially not available 18-
membered ring cyclodepsipeptide 3 has not been prepared so
far, and its stereochemistry had remained unresolved. Here
we unveil a successful total synthesis of chondramide C (3)
that has allowed us to easily access diastereoisomers and
firmly assign the configuration of all its stereogenic centers.[9]
Furthermore, initial biological investigations and results of
computationally docking phalloidin (1) and chondramide C
(3) to its molecular target site on F-actin are reported.[6]
In a retrosynthetic sense it was planned to synthesize 3 via
the peptide acid 4 from acids 6–8, Fmoc-Ala-OH (9), and
[*] Prof. Dr. H. Waldmann, Dr. T.-S. Hu, Dr. S. Renner, S. Menninger,
R. Tannert, Dr. H.-D. Arndt
Max-Planck-Institut für Molekulare Physiologie
Abteilung Chemische Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
Fax: (+49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
hans-dieter.arndt@mpi-dortmund.mpg.de
Scheme 1. Structures of phalloidin (1), jasplakinolide (2), and chondra-
mide C (3), and retrosynthetic disconnection of chondramide C.
Important amino acids are annotated. R=TIPS (triisopropylsilyl);
Fmoc=9-fluorenylmethoxycarbonyl.
and
Technische Universität Dortmund, Fakultät Chemie
Otto-Hahn-Strasse 6, 44221 Dortmund (Germany)
Dr. T. Oda
RIKEN, SPring-8 Center, Structural Physiology Research Group
Kouto 1-1-1, Sayo (Japan)
homoallylic alcohols 5a–d (Scheme 1). For ease of operation
and synthetic flexibility we envisioned assembling the N-
acylated tripeptide 4 on solid support. Ring-closing meta-
thesis (RCM) as the key step could provide the 18-membered
cyclodepsipeptide ring after esterification of the released
peptide acid 4 with alcohols 5a–d. Such a strategy would allow
all building blocks to be varied and pave the way for a
synthesis of a diverse collection of chondramide C ana-
logues.[10] The structural similarity of 3 and 2 as well as
[**] This work was supported by the Max Planck Gesellschaft (H.W.), the
Deutsche Forschungsgemeinschaft (Emmy-Noether grant to
H.D.A.), the state of North-Rhine-Westphalia, and the European
Union (ZACG Dortmund). T.S.H. is grateful to the Alexander von
Humboldt Stiftung for a research fellowship. We thank Prof. Dr. M.
Kalesse for discussions and for making a sample of natural
chondramide C available.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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biosynthetic considerations[11] suggested that the stereogenic
centers in the peptide fragment and C2 might feature the
same absolute configurations. Consequently (R)-b-tyro-
sine,[11] d-N-methyl tryptophan (d-abrine), and l-alanine as
well as S-configured acid 8 were chosen, whereas the
configuration of the two stereocenters of the secondary
alcohols 5 were unclear. Therefore our synthetic work began
with the preparation of all four stereoisomers 5a–d using
Brownꢀs asymmetric crotylboration methodology.[12] O-TIPS-
protected (R)-Fmoc-b-tyrosine 6 was synthesized by a
diastereoselective addition of (S)-N-benzyl-1-phenylethyla-
mine[13] to the corresponding cinnamic acid benzyl ester
followed by hydrogenolysis and Fmoc-introduction (Support-
ing Information).[8e] Fmoc-d-abrine 7 was obtained from d-
tryptophan by two reductive aminations (PhCHO, HCHO)[14]
and protecting group exchange (Supporting Information).
Acid 8 was prepared by stereoselective alkylation using
Seebachꢀs oxazolidinone.[15] The S-configuration of the newly
formed stereocenter of 8 was unambiguously deduced from
an X-ray crystal structure analysis.[16] With all required
building blocks in hand, acid 6 was attached to 2-Cl-trityl
resin to give solid-supported ester 10, which was elongated by
alternating Fmoc deprotection and peptide coupling steps
(Scheme 2). Release of 4 under mildly acidic conditions and
Steglich esterification with alcohols 5a–d in solution afforded
the pure diastereomers 11a–d in excellent overall yields (44–
54% from 10).
The formation of a-branched trisubstituted olefins
embedded in macrocycles by ring-closing metathesis has
previously been found to be challenging and strongly
dependent on structural features of the substrates.[17] After
careful experimentation it was found that treatment of 11 with
25–30 mol% of catalyst 12 in refluxing toluene gave consis-
tent results and delivered the macrocycles 13 in 48–71% yield,
if a constant purging flow of argon was applied throughout.[18]
The cyclodepsipeptides 13 were then easily deprotected to
give the desired final products 14a–d (Scheme 2). The
stereoselectivity of the metathesis reactions depended sig-
nificantly on the configuration of the starting materials. For
example, 11a cyclized to give an inseparable 1.4:1 mixture of
(Z)- and (E)-13a, but macrocycle 13b was formed as the Z
isomer exclusively. The macrocyles 13c and 13d were formed
as pure E isomers (NOE data). Although the reasons for the
observed stereoselectivity currently remain unclear, we note
that the isomer ratios did apparently not change during the
reaction course. We hence assume that the double bond is
formed under kinetic control and that individual geometries
result from distinct ruthenacyclobu-
tane intermediate conformations.[19]
Scheme 2. Preparation of ring-closing metathesis precursors 11a–d and synthesis of chondrami-
Careful analysis of the NMR
spectra and HPLC traces of the
cyclization products 14a–d and com-
parison with data obtained from an
authentic sample[20] clearly revealed
(E)-14c as the naturally occurring
chondramide C (Table 1 and Sup-
porting Information).[9] Furthermore,
the identity was evident from similar
biological activity profiles of authen-
de C and its stereoisomers. a) 2-chlorotrityl chloride resin (1.4 mmolgÀ1), EtN(iPr)2 (4 equiv),
CH2Cl2, RT, 2 h; b) 1. 20% piperidine, DMF (2”20 min); 2. 7 (2.6 equiv), DIC (2.6 equiv), HOBt
(2.6 equiv), DMF, 2.5 h; 3. 20% piperidine, DMF (2”20 min), 4. 9 (2.3 equiv), HATU
(2.3 equiv), HOAt (2.3 equiv), EtN(iPr)2 (4.6 equiv), DMF (2”2.5 h); 5. 20% piperidine, DMF
(2”20 min); 6. 8 (2.3 equiv), HATU (2.3 equiv), HOAt (2.3 equiv), EtN(iPr)2 (4.6 equiv), DMF,
2.5 h; 7. HOAc/trifluoroethanol/CH2Cl2 (1:1:8), 2”1.5 h; c) 5 (3 equiv), EDC (2 equiv), DMAP
(2 equiv), EtN(iPr)2 (2 equiv), CH2Cl2/DMF (20:1), 14 h; d) catalyst 12 (25–30 mol%), Ar
purging, toluene, 1108C, 2 h; e) TBAF (2 equiv), THF, 08C, 1 h. DIC = Diisopropylcarbodii-
mide; DMF = dimethylformamide; HATU = O-(7-azabenzotriazol-1-yl)tetramethyluronium
hexafluorophosphate; EDC = N’-(3-dimethylaminopropyl)-N-ethylcarbodiimide; DMAP = 4-
dimethylaminopyridine; TBAF = tetrabutylammonium fluoride.
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Table 1: Characteristic 1H NMR chemical shifts (at 600 MHz, in ppm) of
compounds 3 and 14a–d, and their effective concentrations against
actin.
Compd
5-H
7-H
3’-
H ’’- 2 H ’’’-H2 ceff [mm][a]
3[b]
(Z/E)-14a
4.71
4.34
5.23
5.50
4.61
0.2
5.00[c]
4.56[d]
4.96
4.56[c]
4.89[d]
4.56
5.17[c]
5.11[d]
5.12
5.46[c]
5.56[d]
5.32
4.69[c]
4.56[d]
4.69
10[e]
(Z)-14b
(E)-14c
(E)-14d
5
0.2
10
4.72
4.67
4.35
4.19
5.23
5.20
5.51
5.48
4.61
4.59
[a] Concentration at which the actin-stabilizing phenotype was fully
developed. [b] Data taken from ref. [5], see Scheme 2 for numbering.
[c] For Z olefin. [d] For E olefin. [e] Apparent ceff of the Z/E mixture.
tic 3 and 14c, whereas the other polyketide stereoisomers
were much less active (vide infra). Therefore the natural
product 3 embodies (R)-b-tyrosine, d-N-methyltryptophan,
l-alanine, and its polyketide configurations are 2S, 6R, and
7R.[21]
Whole-cell microscopy in BSC-1 cells was employed to
evaluate F-actin stabilization properties, using jasplakinolide
(2) as control (Figure 1 and Supporting Information). Cell
shrinkage, reduction or disappearance of F-actin fibers,
formation of large F-actin clumps mainly in the perinuclear
region, and binucleation of cells was prominent for both
synthetic chondramide C (14c) and an authentic sample (3,
data not shown)[20] already at c = 200 nm. This phenotype is in
full accord with earlier studies on actin-stabilizing com-
pounds[4,5] and was indistinguishable from 2 at c = 100 nm
(Figure 1B and E). In contrast, the other isomers studied
(14a, 14b, 14d) induced a comparable phenotype only at c =
5–10 mm (Figure 1C, D, and F), which demonstrates that the
configuration at C7 is a major determinant of the F-actin
stabilizing activity of 3 and of similar importance to the
double bond geometry.
Figure 1. Actin stabilization phenotypes in BSC-1 cells monitored by
whole-cell fluorescence microscopy (magnification 40”) after staining
for actin (red, TRITC-phalloidin, Sigma) and chromatin (blue, DAPI,
Sigma). A) DMSO only (negative control); B) 100 nm jasplakinolide (2,
positive control); C) 10 mm (Z/E)-14a; D) 5 mm (Z)-14b; E) 200 nm
(E)-14c (=3); F) 10 mm (E)-14d. TRITC= tetramethylrhodamin iso-
thiocyanate. DAPI = 4’,6-diamidino-2-phenylindole.
data[6c] were therefore initiated to gain deeper insight into
potential binding modes. The binding of phalloidin (1) to its
target was re-evaluated first.[6a,c,23] The [Ala7]-phalloidin
crystal structure[24] was used as input for a conformational
search in MOE,[25] and the conformational ensemble obtained
in this way was then subjected to unbiased computational
docking onto the actin polymer structural data[6c] using
GOLD.[26]
These results confirm that chondramide C (3 = 14c) is a
competitive ligand for phalloidin (1) on F-actin.[4,5] Com-
pound 1 binds a cavity formed at the point of contact of three
actin protein monomers in the polymeric filament.[6,22]
Molecular docking simulations on the best currently available
Figure 2. Proposed binding modes of [Ala7]-phalloidin and chondramide C on F-actin. A) 3D-orientation of three actin monomers in the F-actin
filament with a binding mode proposition for [Ala7]-phalloidin (yellow) derived from unbiased computational docking experiments. Proposed
hydrogen bonds are indicated by dashed lines. B) Surface representation of F-actin with the binding mode prediction of chondramide C (blue)
overlaid with [Ala7]-phalloidin (yellow).
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Communications
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The top scoring docking results clearly suggested a
binding mode for [Ala7]-1 (Figure 2A), which is characterized
by the indole moiety being in close contact with aromatic
residues (Y198, F200) and cis-Pro(OH)4 pointing towards the
actin R177/D179 salt bridge. 4Hyp4 interacts with S199 by
hydrogen-bonding, and T202 forms a hydrogen bond to the
backbone carbonyl group of Ala1. This model refines earlier
proposals[6a,c,23] and orients phalloidin roughly similar to the
binding mode anticipated by Lorenz et al.[6a] More specifi-
cally, all SAR data available on 1 are in agreement with the
binding geometry found here by unbiased docking.[6c,23,24,27,28]
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The binding of chondramide C (3 = 14c) was investigated
in a similar fashion. Owing to the larger conformational
freedom of 14c, the size of the cavity, and limitations of the
available data (8 Š resolution),[6c] binding modes were
preferred that showed key interactions similar to the phalloi-
din pharmacophore.[29] In our best solution (Figure 1B) the
Trp side chain of 14c similarly interacts with aromatic amino
acid residues, and the Ala in 14c overlays with the respective
Ala5 of 1. The polyketide segment aligns well with the 4Hyp4
and Cys3 of 1, and the Tyr-OH group interacts with T202,
which has been predicted to contribute to binding of 1. In this
binding model of 14c the order of the residues is identical to 1,
despite the opposite Trp stereochemistry in phalloidin (l) and
chondramide C (d). Importantly, the model explains well the
influence of the stereogenic centers in the polyketide back-
bone of 3. Inversion of configuration at C7 attenuated activity
by 100-fold, presumably by populating unfavorable confor-
mations[30] in the peptide segment of chondramide C.
In summary, a total synthesis of chondramide C (3) was
accomplished featuring a rewarding E-selective ring-closing
metathesis as the key step. The excellent overall yield (34%
from 10) highlights the benefit of our swift solid-phase based
synthesis strategy, and chondramide C analogues were rapidly
assembled (19–38% from 10). Phenotypic actin assays
revealed C7 and the double bond in 3 as crucial stereogenic
elements for determining F-actin-stabilizing activity. Compu-
tational docking studies substantiated a pharmacophore
model for phalloidin (1) and provided a binding mode for
chondramide C (3 = 14c). These results are expected to guide
further developments of actin-stabilizing agents in the future.
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Received: March 2, 2008
Published online: July 15, 2008
Keywords: antitumor agents · drug design · metathesis ·
.
natural products · total synthesis
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